Hematopoietic stem cell (HSC) transplantation is widely used to treat a variety of disorders. Despite advances in the use of cord blood and mobilized stem cells, donor material remains limited. This is due to limited stem cells in cord blood, poor mobilization, and the lack of ethnic diversity to provide sufficient matched material. Despite intensive efforts there has been very limited success in generating transplantable HSCs from embryonic stem cells or more recently, from induced pluripotent stem cells. We asked if HSCs could be directly programmed from murine fibroblasts with defined combinations of transcription factors (TFs). A set of 18 candidate TFs was identified and introduced into mouse embryonic fibroblasts (MEFs) containing a reporter with expression specific to primitive endothelial and hematopoietic stem/progenitor cells. Transduction with the 18 TF cocktail resulted in activation of the reporter. Sequential experiments evaluated the requirement of each TF. We identified a set of 4 TFs (Gata2, Gfi1b, cFos, and Etv6) as sufficient for efficient hemogenic induction. These 4 TFs induce a dynamic, multi-stage process that progresses through an endothelial-like intermediate. Transduced MEFs first generate cells with a global endothelial-like gene expression program that are organized into characteristic structures. We show that a Reporter+Sca1+Prominin1+ precursor emerges with hematopoietic activity. Emergent hematopoietic cells have a global gene expression program highly similar to bona fide HSCs most specifically to the specifying HSCs from Aorta Gonad Mesonephros (AGM), placenta, and early fetal liver. We also detect a subpopulation of cells with an HSC cell surface phenotype. Upon transfer of the 4 TFs to inducible vectors where expression can be turned off and the use of aggregation cultures we have been able to demonstrate the development of multi-lineage in vitro colonies. Collectively, our results strongly suggest that the 4 TFs recapitulate a complex developmental program highly similar to fetal hemogenesis. These data are now published in Cell Stem Cell. In this proposal we will further our initial studies with inducible hemogenic programming systems. We will then identify the kinetics of the hemogenic process with a focus on defining the exact temporal requirement for exogenous TF expression. We will employ a broad range of in vitro and in vivo assays. We also propose an in-depth molecular analysis of our hemogenic system emphasizing the generation of intermediate endothelial-like cells and the ultimate emergence of HSC-like populations. The molecular analyses will focus on global mRNA and microRNA expression profiling. Finally, we will build on our preliminary results in the human system. The human studies will closely follow our efforts in the mouse. Collectively, our studies will rigorously establish whether a hemogenic program that yields fully functional HSCs can be specified in vitro and provide a future source of material for regenerative medicine.